Rabies Vaccinology Susan Moore, Rabies Laboratory/KSVDL/College of Veterinary Medicine/Kansas State University, Manhattan, KS 66502, USA
Overview • Human and animal immune responses to rabies vaccines • Responses which are key to mediating or correlating with Rabies protection • Requirements necessary for validating in vitro assays as replacements for in vivo efficacy testing
WHO Immunologic basis for immunization: Module 17 Rabies
Human vaccine response to rabies vaccination - humoral Rupprecht, et. al. Vaccine Volume 27, Issue 51, 27 November 2009, Pages 7141-7148
Human vaccine response to rabies vaccination - cellular Medium PHA Rabies CD4 R-PE FL-2 CD8 R-PE FL-2 Moore, et. al. J Clin Immunol, Vol 26, No 6, November 2006, Pages 533-545.
Peak response Day 3 Day 7 Day 14 Vaccine Year of regimen testing % <0.2 % <0.5 % <0.2 % <0.5 % <0.2 % <0.5 Post- exposure 2010 69.1 91.2 34.0 57.0 11.6 13.0 Post- exposure 2014 57.1 97.4 24.7 85.7 0.0 1.4 Post- exposure 2012 81.2 92.5 64.0 82.1 0.4 1.7
Peak response # subjects Day 3 Day 7 Day 14 post Aoki et al exposure- 0.17 0.30 7.6 IM 10 (0.10-0.42) (0.13-0.42) (2.3-13.0) Comment Comment in discussion post that the peak for those Jones et al exposure- receiving HRIG was at IM 0.17 6.9 day 42, without HRIG 118 nd (0.16-0.19) (5.8-8.1) peaked at day 14 post Jones et al exposure- 0.17 10.3 IM 124 nd (0.16-0.19) (8.8-12.1) Summary of 3 groups, post 0.32 23.2 2 ID and 1 IM, the Briggs et al exposure- (<0.05- (0.4-- lowest responses are ID/IM 154 nd 19.1) 1318.0) im the IM group post Summary of 4 groups, Warrell et al exposure- 0.59 308.72 3 were ID/different # ID 227 nd (0.02-8.39 (5.5-3711.5) sites and 1 IM
Results >0.5 IU/mL >0.1 IU/mL <0.1 IU/mL
Results - % Subjects needing booster 50 50 45 per WHO 0.5 IU/mL per ACIP 0.1 IU/mL 40 40 35 Percent 30 30 Percent 25 20 20 15 10 10 0 5 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 0 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 Year Year A B C D E F A B C D E F The expected percentage of people to require a booster by WHO level is 10-30% after 1 year (Strady, 1998), and by ACIP level is 2- 7% after 2 years (ACIP, 2008).
Animal vaccine response to rabies vaccine • Dog and cat – Aubert, 1992 – Lawson, 1972 – Bunn, 1984 • Hamster, mice • Wildlife Day 28 Day of challenge
Results – Animal studies assay and species difference Open circles: survived Black triangles: succumbed 70% 40% 0.5 IU/mL 0.25 IU/mL Trop. Med. Infect. Dis. 2017, 2, 31; doi:10.3390 http://www.mdpi.com/journal/tropicalmed
Longevity of response • Lawson and Crawley, 1972 – Dogs vaccinated 5 years previously survived challenge – Cats vaccinated 4 years previously survived challenge • Strady, et. al. 1988 – Pre-exposure series and booster at 1 year — human subjects – >95% maintained titers above 0.5 IU/mL for 10 years • What level is significant? 4 weeks after vaccination? Day of challenge? WHO Immunological basis for vaccination Module 17:Rabies, 2017
• The most important role of rabies vaccination is the induction of a sustained antibody response with the help of CD4+ T cell activation. • Protective mechanisms involved in the immune response to rabies infection indicate cooperative action of neutralizing antibody, cellular immune soluble factors as well as action by CD8+ T cells to play primary roles. • “Early/high and late/low” responders to rabies vaccination have been noted with modern cell culture vaccines. Rabies 3 rd Edition, 2013 Chapter 12, page 463
Correlates of Protection • Rabies Virus Neutralizing Antibodies • RFFIT and derivatives (e.g. FAVN, EP) • Issues – Challenge strain – Virus dose – Standard reference serum – Tissue culture cells – Calculation (ED50, IU/mL) • Rabies anti-glycoprotein antibodies – Measured by ELISA • Other assays
Challenge studies: rabies antibody level in vaccinated animals and survival • Animals with “detectable” RVNA at day of challenge survive? – Mostly….. – Rare reports of animals with levels above 0.5 IU/mL dying after challenge • Animals with no detectable RVNA at day of challenge succumb? – Some do, some survive – Cellular immunity or undetectable antibody
0.5 IU/mL – what does it mean? Rev Sci Tech. 1992 Sep;11(3):735-60. Practical significance of rabies antibodies in cats and dogs. Aubert MF 1 . 1 Centre national d'études vétérinaires et alimentaires, Laboratoire d'études sur la rage et la pathologie des animaux sauvages, Malzéville, France. Abstract Doubt has sometimes been cast upon the protective effect of rabies antibodies in serum. Animals and humans suffering from fatal rabies often produce high antibody titres, while rabies cases are also observed in vaccinated animals. Cellular immunity is also largely involved in protection. Nevertheless, a large number of laboratory experiments and field observations clearly demonstrate that cats and dogs which develop antibodies after vaccination and before challenge have a very high probability of surviving any challenge, no matter how strong the dose and which virus strain was used. Rabies antibody titration can, therefore, afford a strong additional guarantee to the vaccination certificates accompanying domestic carnivores during transportation between countries. Quarantine rules should also be adapted to the epidemiological features in the exporting country, e.g. statistics of vaccination failure in cats and dogs and host-virus adaptation of the rabies strains circulating in these countries. PMID: 1472723 [PubMed - indexed for MEDLINE]
Human Minimum Acceptable RVNA level • Based on early vaccine clinical trials – Measurement of RVNA by mouse neutralization test (MNT) or Rapid Fluorescent Focus Inhibition Test (RFFIT) – Determination of adequate vaccine response • Two guidelines give recommendations: – World Health Organization (WHO) – 0.5 IU/mL – Advisory Committee on Immunization Practices (ACIP) – complete neutralization of rabies virus at a 1:5 serum dilution in the RFFIT (0.1 IU/mL) – These two levels are different
CFR 113.209 Immunogenicity • Serology required on days 30, 90, 180, 270 & 365 • Species other than carnivores: • Challenge can be limited to: – 5 vaccinates with the lowest day 270 SN titers, plus – 5 vaccinates w/lowest day 365 SN titer, and – All vaccinates with SN titers <1:16 by RFFIT, and – 5 controls • Valid test requires: – All vaccinates must survive – 80% of controls must die due to rabies (humane endpoints allowed)
Proposed in vitro assay replacement for NIH: Serology • OIE – for veterinary vaccines allow for potency test by challenge or serological method. • Krämer, et al., 2009 – Modification of the RFFIT/FAVN following the European Pharmacopeia method for RIG potency for higher precision
Proposed in vitro assay replacement for NIH: Antigen Quantification • Gilbert et. al., 2013 – Immuno-capture ELISA using one monoclonal to antigenic site III for both capture and detection • Chabaud-Riou et. Al., 2017 – Immuno-capture ELISA using two monoclonals : to antigenic site II for capture and to antigenic site III for detection.
Replacing the NIH test • There are clear advantages in replacement of the NIH test for rabies vaccine (cost, time, scientific validity, and 3R’s • Validation of a new method is a concern – correlation to NIH is expected to be poor • Reluctance to give up the NIH test – It has been the ‘gold - standard’ for over 60 years – Accepted globally – Risk aversion (sub potent lots) – Availability of new method reagents and consensus of method • Better acceptance of a combination of serological and antigen quantification results for vaccine approval
Validation of assays ICH Expert Working Group(Quality) ICH Topic Q 2 (R1) Validation of Analytical Procedures: Text and Methodology 2018 WHO Expert Committee on Biological Standardization Sixty – seventh report, TRS 1004, 2017. FDA (2018) Guidance for Industry Bioanalytical Method Validation. 1-41. “Validation of an analytical method is the process by which it is established, by lab o ratory studies , the performance characteristics of the method meet the requirements for the intended analytical application .” – United States Pharmacopoeia General Chapter 1225. Good fit?
Assay Validation – Parameters as appropriate to the method ▪ Specificity ▪ Linearity ▪ Range ▪ Accuracy ▪ Precision ▪ Repeatability ▪ Intermediate precision ▪ Detection limit ▪ Quantitation limit/Sensitivity ▪ Robustness ▪ Stability ▪ Dilution effects
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